The present invention relates to the preparation and use of formalin-killed colonization-factor-antigen (CFA)-expressing E. coli organisms for vaccination against enteric infection/diarrhea caused by enterotoxigenic E. coli bacteria in humans.
Specially, the invention relates to a method of producing a vaccine composition against enteric infection caused by enterotoxigenic E. coli bacteria in humans, and a method of preventing an enteric infection caused by enterotoxigenic E. coli bacteria in humans.
Diarrhea caused by enterotoxinogenic Escherichia coli (ETEC) is an important health problem, particularly in developing countries and in travellers to these areas. In hospital- and clinic-based studies of acute diarrhea in developing countries ETEC has been identified in 10-50% of the cases, the average being ca 20% in children less than 5 years, and slightly higher in older age groups. Likewise, ETEC has been identified as the causative agent in at least one third to one half of cases of acute diarrhea among persons travelling from industrialized to developing countries.
The illness caused by ETEC ranges from mild diarrhea without dehydration to cholera-like disease. In the first 5 years of life many children in developing countries suffer from 1-2 episodes of diarrhea caused by ETEC each year. Although in the majority of ETEC infections symptoms are relatively mild, ETEC accounts for more than one billion diarrheal episodes and one million deaths annually among children in developing countries. Thus, any interventions that could reduce ETEC mortality and morbidity even partially might be of great public health significance.
No vaccine for use in humans against ETEC diarrhea is yet available. However, in a large field trial of a newly developed oral cholera vaccine it was found that the B subunit component of this vaccine, which cross-reacts immunologically with the heat-labile enterotoxin (LT) of ETEC, afforded significant protection not only against cholera but also against diarrhea caused by LT-producing ETEC. The protection against ETEC infection was particularly pronounced against illness associated with severe, life-threatening dehydration which was reduced by 86% by the vaccine during the first three months after immunization.
MECHANISMS OF DISEASE AND IMMUNITY. To cause disease, ETEC must be able to colonize the small intestine, and to elaborate LT and/or a heat-stable enterotoxin (ST, STa). E. coli LT is similar to cholera toxin in structure and function, consisting of a toxin-active A subunit attached to five B subunits that mediate binding to cell membrane receptors. The ST molecule is a small polypeptide of only 19 amino acid residues, that stimulates guanylate cyclase activity in intestinal cells. Different from LT which is a strong immunogen, ST is non-immunogenic unless experimentally conjugated to a larger carrier protein. ST-only and LT/ST-producing strains are important causes of diarrhea in endemic areas, whereas LT-only strains frequently cause disease in travellers to developing countries. The proportion of ETEC strains with different enterotoxin profiles varies from country to country.
In many ETEC strains adhesion to intestinal mucosa is mediated by antigenically distinct fimbriae. In strains pathogenic for man three main adhesins have been identified; they are referred to as colonization factor antigens CFA/I, CFA/II, and CFA/IV (formerly called PCF8775). CFA/I is a single homogenous fimbrial antigen whereas CFA/II comprises the coli surface (CS) antigens CS1, CS2 and CS3, and CFA/IV the CS4, CS5 and C6 antigens. Although the prevalence of these different colonization factors varies geographically, CFA/I, CFA/II or CFA/IV are usually found on one-half to three-quarters of ETEC isolated from cases with clinically significant diarrhea. However, additional adhesins are also likely to be identified.
The highest ETEC infection rates in endemic areas are seen in young children. The findings of a decreased attack rate with increasing age and of a higher proportion of asymptomatic cases in adults than in children suggest that naturally acquired protective immunity may develop. Similarly, a degree of resistance against ETEC diarrhea develops among travellers during prolonged residence in high-risk countries. Experimental studies in animals and human volunteers have also shown that ETEC infection may give rise to substantial immunity against rechallenge with the homologous organisms.
There is evidence that both antibacterial and antitoxic immunity contributes to protection against ETEC diarrhea. Antibacterial immunity against ETEC may to a large extent be ascribed to immunity against the different colonization factor antigens (CFA), even though antibodies against O-antigen may play a role as well for protection against ETEC of homologous O-gruoups. In animals, anti-CFA antibodies have protected against challenge with ETEC expressing the homologous CFAS. similarly, in both animals and human volunteers oral or intraintestinal immunization with ETEC strains expressing CFA/I, CFA/II or CFA/IV has induced protective immunity against subsequent challenge with E. coli carrying the homologous CFA/CS-factor.
Naturally induced antitoxic ETEC immunity is only directed against LT since native ST is not immunogenic. The anti-LT immune response is mainly against the B subunit portion of the molecule, which cross-reacts immunologically with the B subunits of cholera toxin. This explains why, as mentioned, oral immunization with cholera B subunit could induce protection against ETEC diarrhea [1]; interestingly protection was induced not only against LT-only but also against LT/ST ETEC strains [1]; a finding also supported by findings in animals after immunization with either cholera or LT B subunits (unpublished data).
It is also known that in humans clinical ETEC disease evokes significant antitoxic as well as antibacterial immune responses in the intestine resulting in specific IgA antibody titer increases in intestinal lavage fluid against LT and homologous CFA and O antigens [2]. Both anti-enterotoxin and anti-colonization factor antibodies can independent of each other protect against experimental ETEC infection and, when being present together in the intestine, these antibody specificities have been found to cooperate synergistically in protecting against ETEC disease [3].
In contrast to the well-established protective function of anti-LT immunity against ETEC disease, the significance of anti-ST immunity for protection remains undefined. Although ST in its natural state is not immunogenic it may give rise to ST-neutralizing antibodies when being used coupled to a carrier protein. This suggests that also vaccine-induced anti-ST immunity may be an attainable goal. However, different ST-carrier conjugates tested to date, derived either by chemical coupling or recombinant DNA techniques, have all retained significant, though sometimes reduced toxic activity. Therefore, several synthetic modified ST-peptides have been prepared recently in an attempt to identify nontoxic ST-related epitopes. Synthetic oligonucleotides encoding for similar peptides have also been made and fused to the gene for cholera B-subunit and when inserted into Vibrio cholerae these chimeric genes encode production of high concentrations of completely nontoxic ST-B subunit fusion protein. Immunization of experimental animals with such chemically derived or genetically engineered nontoxic peptide-B-subunit conjugates has evoked anti-ST antibody responses but so far with only weak neutralizing activity.
CANDIDATE VACCINES. Based on this knowledge about the key protective antigens of ETEC bacteria and of the main immune mechanisms operating against ETEC infections, it may be concluded that an effective ETEC vaccine should be given orally and ideally evoke both anti-colonization and antitoxic immune responses in the intestine. Thus, the vaccine should contain a combination of bacterial cell- and toxin-derived antigens. Different live or inactivated ETEC vaccine candidates have recently been considered based on these premises.
Live vaccines. Live bacteria expressing the major CFAs and producing B subunit or a related enterotoxoid may be considered since such a vaccine through multiplication in the gut might provide a sustained antigen stimulation of the local intestinal immune system. However, since the different colonization factors are normally not expressed on the same strains and it has not been possible to clone the genes for different CFAs into the same host organism, such vaccines mustxe2x80x94at least for the present momentxe2x80x94be based on a mixture of several different strains. There are several problems, however, with this kind of vaccine approach. Among these are the risk for overgrowth of one of the included vaccine strains with suppression of the others; reversion to toxicity by uptake of toxin-encoding plasmids; low production of enterotoxoid during growth in vivo; and poor survival of the vaccine strains during storage. Therefore live oral ETEC vaccines are not yet in sight.
Non-living vaccines. The most important of the somatic antigens to be included in a vaccine are those CFAs that have a high prevalence on ETEC strains in different geographic areas. These antigens include CFA/I, CFA/II and CFA/IV and possibly a few additional CFAs as yet to be defined. However, purified CFA antigens may be relatively expensive to prepare and, furthermore, after oral administration isolated CFAs have proved to be sensitive to degradation in the human gastrointestinal tract [4, 5].
A more practical way to construct a vaccine may be to prepare killed ETEC bacteria that express the most important CFAs on their surface and combine these organisms with an appropriate toxoid component. Important vaccine preparation problems to overcome would be to (a) find a procedure that would safely kill the vaccine strains while still preserving the antigenic and adhesive (hemagglutinating) properties of different CFAs (ideally this procedure should also stabilize the CFAs against degradation in the human intestinal milieu), and (b) find conditions allowing high-level expression of CFAs on bacteria during growth in liquid medium in a fermentor in order to facilitate large-scale vaccine production.
A well-known and therefore advantageous method to inactivate bacteria for use as whole cell vaccines would be the use of formalin treatment. Usually a formalin concentration of 1 M is used for such purposes. We found that treatment of CFA expressing ETEC with commonly used vaccine preparation methods safely killed the ETEC organisms but that it at the same time destroyed most or all of the CFA antigen content and therefore these methods were not well suited for preparation of inactivated ETEC strains with retained CFA immunogenicity. In accordance with this, Levine [6] has described the limited success of Tacket et al. to use formalin-treated ETEC organisms of one strain for stimulating IgA antibody formation and protection in human volunteers. The vaccine was prepared from E. coli strain E1392-75-2A, an O6:H16 biotype A strain that expresses CS1 and CS3 fimbriae, but does not elaborate LT or ST. When used previously as a live oral vaccine, a single dose of E1392-75-2A provided significant protection to volunteers against experimental challenge with an LT+/ST+ ETEC strain of serotype 0139:H28 that expresses CS1 and CS3. (The E1392-75-2A bacteria were formalin-inactivated by investigators at the U.S. Army Vaccine Production Facility of the Walter Reed Army Institute of Research at Forest Glen, Md.). Three doses of vaccine (each containing 5xc3x971010 inactivated bacteria) with NaHCO3 were given by Tacket et al. at two-week intervals to nine volunteers. Significant rises in CFA antibody were detected in serum in two of nine vaccinees, whereas four of nine had detectable rises in intestinal SIgA anti-fimbrial antibody. A small challenge study was carried out in which four of the vaccinees and ten controls were challenged with pathogenic ETEC strain E24377A (0139:H28, CS1, CS3, LT+/ST+). No evidence of protection was detected: diarrhea occurred in two of four vaccinees and in six of ten controls.
With regard to expression of CFAs in liquid medium this has not been reported. Indeed, until now such expression has not been regarded possible since liquid medium is known to suppress expression of CFAs and instead induce expression of mannose-sensitive type 1 pili [7].
Initial efforts to develop prototype vaccines based on ETEC expressing different CFAs and Cs proteins [8] had to rely on growing the organisms on solid agar plates which seriously impedes the possibility for larger-scale vaccine production. In the present invention we have now developed a procedure which allows equally good expression of CFAs on ETEC during growth in liquid medium in shake culture as under optimal CFA-promoting agar plate growth conditions and we also show high-level expression of CFAs on E. coli during fermentor cultivation conditions. We also describe a procedure by which these liquid medium fermentor-grown E. coli are inactivated with formalin in a way which ensures safe killing of the tested ETEC vaccine strains and at the same time both preserves and stabilizes the CFAs on the bacterial surface in a defined quantifiable manner.